Atherosclerosis, the most common form of arteriosclerosis, is a disease of large and medium-sized arteries (e.g., coronary, carotid, and lower extremity arteries), and of the elastic arteries, such as the aorta and iliac vessels. The atheroma, or fibrofatty plaque within the intima that consists of a lipid core and fibrous cap, is pathognomonic (Robbins Pathologic Basis of Disease 557 (Cotran et al. eds., 4th ed. 1989)). In addition to being a primary risk factor for myocardial and cerebral infarcts, atherosclerosis is responsible for such medical conditions as chronic lower extremity ischemia and gangrene, and for mesenteric occlusion. Despite a recent reduction in mortality from coronary heart disease, about 50% of all deaths in the United States are still attributed to atherosclerosis (Scientific American Medicine §1 (Rubenstein et al. eds., 1991)).
Epidemiologic, postmortem, and angiographic studies have firmly established a causal relationship between elevated serum cholesterol levels and the genesis of atherosclerosis (Levine et al., Cholesterol Reduction In Cardiovascular Disease, N Eng J Med 332(8):512-521 (1995)). Although there is no single level of plasma cholesterol that identifies those at risk, in general, the higher the level, the higher the risk. However, the risk rises significantly with cholesterol levels above 200 mg/dl (Robbins Pathologic Basis of Disease, supra, at 559). Levels of total cholesterol are typically classified as being desirable (<200 mg/dl), borderline high (200-239 mg/dl), or high (≧240 mg/dl). Dietary treatment is usually recommended for patients with high risk levels of low density lipoprotein (LDL) cholesterol and for those with borderline-high risk levels who have at least 2 additional risk factors for atherosclerosis (e.g., hypertension, diabetes mellitus, cigarette smoking, etc.). However, dietary therapy has been found to be effective only in patients whose diets were higher than average in cholesterol and saturated fats (Adult Treatment Panel II. National Cholesterol Education Program: Second Report of the Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, Circulation 89:1333-1445 (1994)), and would be ineffective in patients with a genetic predisposition to hypercholesterolemia. In the case of persistent high cholesterol levels, drug therapy may be prescribed.
Currently marketed drugs for the treatment of hypercholesterolemia work by such methods as inhibiting de novo cholesterol synthesis and/or stimulating clearance of LDL cholesterol by the LDL receptor (e.g., lovastatin), decreasing the production of very low density lipoprotein (VLDL) (e.g., gemfibrozil), or by inhibiting bile acid reabsorption in the intestines (e.g., cholestyramine). Examination of cholesterol metabolism, however, also reveals that the process of reverse cholesterol transport allows a pathway through which cholesterol may be removed from tissues and may exit the body. At present, there is no known method for measuring the rate of cholesterol flow through the reverse cholesterol transport pathway from tissue to excretion in a living organism.
Reverse cholesterol transport (RCT) is a biological pathway through which cholesterol is mobilized and transported from the peripheral tissues of the body to the liver. As shown in FIG. 1, there are two arms of the pathway, represented by efflux of cholesterol from extrahepatic tissues (the high density lipoprotein (HDL) arm or first arm of RCT) and transport of cholesterol from the bloodstream to the liver (post-HDL arm or second arm of RCT). Eventually, cholesterol is excreted into the bile, and then ultimately, from the body. RCT represents the only known biological pathway or active mechanism by which cholesterol can be removed from tissues. As mentioned above, because of the well-established role of cholesterol in atherogenesis, RCT is considered a key anti-atherogenic process and is generally believed to be the explanation for anti-atherogenic properties and clinical correlation with reduced cardiovascular risk of the high density lipoprotein (HDL) fraction of plasma.
However, HDL levels are now recognized to reflect only one component of the molecular pathway of RCT (FIG. 1), and do not necessarily reflect the true flow of cholesterol through the RCT pathway. The RCT pathway involves the transport of cholesterol from extrahepatic tissues into plasma by HDL, then delivery to IDL (intermediate density lipoprotein) via the action of lecithin-cholesterol acyl transferase (LCAT), and then eventually to LDL (low density lipoprotein). Thereafter, some of the LDL is taken up by the liver and excreted as bile acids into the intestines (Ganong W. F., Review of Medical Physiology 284-288 (15th ed. 1991)). Other pathways of RCT have been considered but the above-noted sequence is currently believed to most likely predominate.
The molecular details of the RCT pathway have come into increasing focus in the past several years. One important implication of these recent advances in molecular understanding is the recognition that plasma HDLc (HDL-cholesterol) levels in isolation may or may not reflect true flux through the pathway, depending upon the underlying mechanism responsible for the change in HDLc. For example, if the plasma concentration of HDLc in an individual represents flux from tissues through ABC(A)-1 (the ATP-binding cassette transporter) into plasma apoAI-containing particles, as in ABC(A)-I heterozygotes, then HDLc is a useful marker. However, if HDLc in another individual accumulates because of inhibition of delivery of HDLc to its acceptors (e.g., due to reduced cholesterol ester transfer protein activity, reduced hepatic SRBI (scavenger-receptor BI) activity), then HDLc levels will not reflect RCT. The situation can be particularly complex, when considering the impact on RCT of interventions that alter the production and fate of apoB containing particles, such as the statins. Because apoB particles are capable of carrying cholesterol forward (i.e., to the tissues) as well as in reverse (i.e., back to the liver), the actual fate of apoB particles in an individual may contribute to the efficiency of RCT at any plasma HDL level. The possibility of a dissociation between HDLc concentrations and RCT is thereby raised in the setting of effective statin therapy (or any other intervention that promotes return of VLDL and LDL particles to the liver).
Measuring the rate of a biochemical process such as RCT is more difficult than measuring the concentration of biochemical molecules. The former requires kinetic methods, while the latter involves static measurements. Kinetic measurements must include the dimension of time (i.e., a timed procedure must be performed), because all rates include time in the denominator (e.g., mg/min for biochemical rates, analogous to miles/hour for physical rates of motion). Typically, for biochemical kinetics, the molecule of interest or a precursor to it is labeled, and the flow of the label from the labeled molecule into various routes is measured over time.
Theoretically, tissue cholesterol may be labeled to follow its efflux from peripheral cells, but in practice, it has been essentially impossible to label non-hepatic cholesterol without labeling hepatic and blood cholesterol at the same time, whether the labeled material administered is cholesterol itself or its biosynthetic precursors (e.g., 14C-acetate, 3H2O, or 2H2O).
This is the case for several reasons: 1) hepatic cholesterol synthesis is very active, so that standard labeled biosynthetic precursors for cholesterol in the body will unavoidably label cholesterol in the liver; 2) there are no known labeled substrates that target peripheral (non-hepatic) tissues exclusively; 3) the cholesterol pools in peripheral tissues are very large and slow to turn over, so it takes a long time (weeks or months) to achieve adequate labeling; and 4) cholesterol exchanges rapidly between liver, blood and tissues, so that it quickly becomes impossible to distinguish the origin of labeled cholesterol and to infer directional rates of transfer.
For all these reasons, an in vivo method for measuring the rate of reverse cholesterol transport is needed and would have great utility for medical care and drug discovery and development.